Characterization of an early-diverging KCNE potassium-channel auxiliary subunit in the jawless vertebrate lamprey

Figures

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  Figure 1.. Sequence, genomic context, and transcript distribution of lamprey KCNE0. (A–C) Amino acid sequence alignment (A), phylogenetic tree (B), and percent identity (C) of lamprey and human KCNE subunits. Alignments were generated with Clustal Omega (Madeira et al., 2024) and displayed using ESPript3 (Robert and Gouet, 2014). Residues corresponding to the ‘triplet’ (Melman et al., 2001; Melman et al., 2002) motif are highlighted with an orange square. (D) Genomic region containing the kcne locus in sea lamprey, shown from the Ensembl genome browser (Martin et al., 2023). For sequence comparison, KCNE0 from lamprey species (sea lamprey, PmKCNE0; NCBI Accession Number XP_032831116.1; Far Eastern brook lamprey, LrKCNE0; XP_061431545.1 and Arctic lamprey, LcKCNE0; see Figure 1—figure supplement 2), the five human KCNE subunits (HsKCNE1, NP_000210.2; HsKCNE2, NP_751951.1; HsKCNE3, NP_005463.1; HsKCNE4, NP_542402.4; and HsKCNE5, NP_036414.1) and zebrafish KCNE6 (DrKCNE6) (Kasuya et al., 2024) were used. (E) RNA-seq-based transcript levels of KCNQ1 (XM_061564454.1) and KCNE0 (XM_061575561.1) from 10 organs of Far Eastern brook lamprey quantified using a decoy-aware Salmon index. Values are…

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  Figure 1—figure supplement 1.. Open reading frame (ORF) nucleotide and corresponding amino acid sequence of KCNQ1 in Arctic lamprey. The ORF nucleotide sequence and the translated amino acid sequence of Arctic lamprey KCNQ1 used for cloning and electrophysiological recordings are shown. The primer-binding sites used for RT-PCR analysis in Figure 1F are indicated with orange arrows.

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  Figure 1—figure supplement 2.. Open reading frame (ORF) nucleotide and corresponding amino acid sequence of KCNE0 in Arctic lamprey. The ORF nucleotide sequence and the translated amino acid sequence of Arctic lamprey KCNE0 used for cloning and electrophysiological recordings are shown. The primer-binding sites used for RT-PCR analysis in Figure 1F are indicated with orange arrows.

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  Figure 2.. Biophysical properties of lamprey KCNQ1 and modulation by KCNE0. (A–G) Representative current traces (A–F) and conductance–voltage (G–V) relationships of lamprey KCNQ1 WT expressed alone or co-expressed with the corresponding KCNE0 WT. (H–J) Representative ionic-current and fluorescence traces (H, I) and fluorescence–voltage (F–V) relationship (J) of PmKCNQ1vcf WT expressed alone or co-expressed with PmKCNE0 WT. Error bars denote mean ± s.e.m. for n=5 in (G, J). Statistical comparisons of G–V V1/2 values for lamprey KCNQ1 WT expressed alone are provided in Supplementary file 3. V1/2 values were not determined for KCNQ1–KCNE0 complexes because the G–V relationships were nearly saturated over the tested voltage range. Source data are provided in Source data 1.

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  Figure 2—figure supplement 1.. Amino acid sequence alignment of KCNQ1 from various species. Amino acid sequence alignment of KCNQ1 from human, zebrafish, lampreys, and vase tunicate generated with Clustal Omega (Madeira et al., 2024) and displayed with ESPript3 (Robert and Gouet, 2014). The residue corresponding to the labeling site in human KCNQ1 (G219) (Osteen et al., 2010; Osteen et al., 2012) is highlighted with an orange circle. For sequence alignment, human (HsKCNQ1; NCBI Accession Number NP_000209.2), zebrafish (DrKCNQ1; NP_001116714.1), sea lamprey (PmKCNQ1; XP_075921450.1), Far Eastern brook lamprey (LrKCNQ1; XP_061420438.1), Arctic lamprey (LcKCNQ1; see Figure 1—figure supplement 1), and vase tunicate (CiKCNQ1; NP_001153537.1) KCNQ1 were used.

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  Figure 3.. Regions of KCNE0 important for KCNQ1 modulation. (A) Schematic diagram of N- and C-terminal truncation constructs of PmKCNE0. (B–F) Representative current traces (B–E) and conductance–voltage (G–V) relationship (F) of PmKCNQ1 WT expressed alone or co-expressed with each of the N-terminally truncated PmKCNE0 constructs. (G–N) Representative current traces (G–M) and G–V relationship (N) of PmKCNQ1 WT expressed alone or co-expressed with each of the C-terminally truncated PmKCNE0 constructs. Error bars denote mean ± s.e.m. for n=5 in (F, N). (O–T) Confocal images of oocytes expressing C-terminally eGFP-tagged PmKCNE0 WT or truncation constructs, and an uninjected oocyte (control). Source data are provided in Source data 1.

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  Figure 4.. Species dependence of KCNQ1–KCNE compatibility. (A–C) Representative current traces (A, B) and conductance–voltage (G–V) relationship (C) of HsKCNQ1 WT expressed alone or co-expressed with PmKCNE0 WT. (D–F) Representative current traces (D, E) and G–V relationship (F) of DrKCNQ1 WT expressed alone or co-expressed with PmKCNE0 WT. (G–I) Representative current traces (G, H) and G–V relationship (I) of CiKCNQ1 WT expressed alone or co-expressed with PmKCNE0 WT. (J–L) Representative current traces (J, K) and G–V relationship (L) of PmKCNQ1 WT co-expressed with HsKCNE1 WT or HsKCNE3 WT. Error bars denote mean ± s.e.m. for n=5 in (C, F, I, L). Source data are provided in Source data 1.

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  Figure 5.. Intracellular leucine substitutions shift KCNE0 modulation toward a KCNE4-like inhibitory effect. (A) Close-up view of the interface between KCNQ1 and KCNE0 within the PmKCNQ1–PmKCNE0–PmCaM complex structure generated by SwissModel server (Waterhouse et al., 2018). The model was built with amino acid sequences of PmKCNQ1 (XP_075921450.1), PmKCNE0 (XP_032831116.1), and PmCaM (XP_032811771.1), using the human KCNQ1–KCNE3–CaM structure (PDB: 6V00) as a template. Single KCNQ1, KCNE0, and CaM subunits are shown in blue, red, and green, respectively. The other regions are shown in gray. Residues used for motif-based mutagenesis are shown as sticks. Molecular graphics were prepared with CueMol (http://www.cuemol.org/). (B) Sequence alignment around the intracellular region corresponding to the KCNE4-related juxtamembrane tetra-leucine motif (Ciampa et al., 2011). (C–F) Representative current traces (C,D), conductance–voltage (G–V) relationship (E), and current amplitude at +60 mV (F) of PmKCNQ1 WT co-expressed with PmKCNE0 WT, PmKCNE0 H75L, or PmKCNE0 P73L/F74L/H75L. In (E), error bars denote mean ± s.e.m. for n=5. In (F), error bars denote mean ± s.e.m. for n=8. Statistical…

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  Figure 5—figure supplement 1.. Attempted conversion of KCNE0 toward a KCNE1-like effect. (A) Close-up view of the interface between KCNQ1 and KCNE0 within the PmKCNQ1–PmKCNE0–PmCaM complex corresponding to the structure shown in Figure 5A. (B) Sequence alignment around the KCNE transmembrane segments highlighting the ‘triplet’ region (Melman et al., 2001; Melman et al., 2002). (C, D) A representative current trace (C) and conductance–voltage (G–V) relationship (D) of PmKCNQ1 WT co-expressed with the HsKCNE1-like triplet mutant of PmKCNE0 (PmKCNE0 L54T). In (D), error bars denote mean ± s.e.m. for n=5. Source data are provided in Source data 1.